How to calculate the malondialdehyde (MDA) concentration for analyzing lipid peroxidation in plants under abiotic stress?
Estimation of lipid peroxidation was assayed spectrophotometrically using thiobarbituric acid-malondialdehyde assay (TBA-MDA). Lipid peroxides were extracted using 0.5 g fresh tissue with 0.3 ml of 1% meta-phosphoric acid (pH 2) and 1 ml of 0.6% thiobarbituric acid (TBA) in air tight tube and kept in a boiling water bath for 45 min. The samples were cooled at room temperature and 5 ml butanol was added along with stirring of mixture. The butanol phase was separated by centrifugation at 1000 rpm for 10 min and transferred to glass cuvettes. The color of TBA chromogen was measured at 532 nm (HODGES &al. 1999).
MDA level = [OD532 x 100/1.56] x TV/ [d wt x 1000]
OD = optical density; TV = total volume of the extract (ml); d wt = weight of the dry leaf tissue (g)
thanks Heshmat! i have one more question. i have used different protocol, can i use this above stated formula for the calculation of MDA?
i have used this protocol
Measurement of lipid peroxidation, concentration of Malondialdehyde (MDA) was taken according to Dhindsa et al. 1981 with some modification. 0.3 gms of frozen plant material was homogenized with 0.5% TCA (trichloro acetic acid) and then centrifuged at 5000 rpm for 15 mins. The mixture containing 1 ml supernatant obtained above was added to 3 ml of 0.5% TBA (thio barbituric acid) in 20% TCA was heated at 100°C for 30 mins in a water bath and then cooled to room temperature. The absorbance was taken at 532 nm and 600 nm.
Yes, you can use the above stated formula for the calculation of MDA
I think same formula can be used. But instead of dry weight use wet liver tissue.
Hello heshmat ,
if we are using different protocol,is it good to apply the same formula..
And can you provide reference paper for that which is accessible.
hello heshmat sir,
TV - volume of extract
this is ..volume of extract taken in cuvette (3ML) or volume of starting material+ 1o ml TCA
i AM HAVING HIGH VALUE OF TBARS IN SEVERE WATER STRESS AS COMPARE TO MODERATE STRESS..WHILE IN CONTROL THIS IS HIGHEST..WHAT DOES IT INDICATE?
Dear Sonam Sneha,
I got the answer from an expert in Delhi.
(Absorbance at 532) - (Absorbance at 600) is Absorbance due to MDA-TBA abduct.
Extinction coefficient of this MDA-TBA abduct at 532 nm is 155 mM-1cm-1.
Concentration of MDA (mM) = (A532 - A600)/155
Let us presume
(i) A532 is 0.75
(ii) A600 is 0.05
(iii) Volume of reaction mixture is 2 ml (one ml sample + one ml 0.5% TBA in 20% TCA)
(iv) path length is one cm
Concentration of MDA (mM) = (0.75 - 0.05)/155 = 0.6/155 = 0.00387
Concentration of MDA (microM)= 0.00387 x 1000 = 3.87
In other words one microM (means one micromole in 1000 ml) has a value of 3.87. Therefore, one ml reaction mixture will have 3.87/1000 micromole of MDA (= 3.87 nmoles). If reaction mixture is two ml then MDA present in two ml will be 3.87 x 2 = 7.74 nmoles.
Depending on the qualtity of material (irrespective of plant/animal/microbe) you had taken and the quantity of (-%)TCA (or some other solvent) you used for extracting MDA, you need to calculate actual MDA content (either per g fresh or dry weight or some other parameter).
https://www.researchgate.net/post/How_can_we_calculate_the_malondialdehyde_MDA_contents
Hope this help.
I think this link is useful,
http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/
According this procedure : Please tell me about the number of 6.45 and 0.56 and the unit of concentration of MDA.
Determinations of membrane oxidation
MDA concentration was measured according to Dhindsa et al.
(1981) with modification. Tissue samples (2.0 g) were ground in
liquid nitrogen and extracted in 5mL 10% (w/v) trichloroacetic acid
(TCA). After centrifugation at 10,000×g for 15 min, 2mL aliquot of
the supernatant was mixed with 2mL 10% (w/v) TCA containing
0.6% (w/v) thiobarbituric acid (TBA). The mixture was heated to
100 ◦C for 20 min, quickly cooled and centrifuged at 10,000×g for
10 min. The supernatant was collected and absorbances at 532, 600
and 450nm were then measured in spectrophotometer (Shimadzu
UV-1750). The MDA concentration was calculated according to
the formula: 6.45×(A532 −A600)−0.56×A450.
Postharvest Biology and Technology 62 (2011) 115–120 .
OD600 values are subtracted from the MDA-TBA complex values at 532 nm and MDA concentration is calculated using the Lambert-Beer law with an extinction coeeficient εΜ= 155 mM-1cm-1. Results are presented as μmols MDA g-1FW.
The link
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC35156/
For this publication : Oxidative Damage in Pea Plants Exposed to Water Deficit or Paraquat
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC35156/
by I Iturbe-Ormaetxe - 1998 - Cited by 514
Article Oxidative Damage in Pea Plants Exposed to Water Deficit or Paraquat
How to calculate the %age of MDA ?
As level of MDA increased and Normal value is less then how to caculate %age ?
What if the calculation of
MDA (mM) = (A532 - A600)/155
is negative? is it wrong?
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